RESPONSIVENESS OF GNRH RECEPTOR SIGNALING

拟议研究的长期目标是调查 GnRH受体信号的反应性。健康的重要性 这些研究表明，GnRH作为一种治疗方法的机制， 前列腺癌是基于其倾向于脱敏LH分泌 从而减少睾丸激素的分泌细胞内蛋白质 已经受到审查的潜在监管机构 GnRH受体信号传导的反应性是GRK家族的成员（G 蛋白偶联受体激酶）和β抑制蛋白1和2。GRK 2，3， 和5以及β抑制蛋白1和2在大鼠中天然表达 垂体前叶及其在GnRH中的实验表达 表达受体的COS-1细胞抑制GnRH刺激的 IP3是由GnRH引起的第二信使级联中的一员。发展 在大鼠中表达GRKs的创新腺病毒方法 垂体前叶细胞已经显示出它们具有强烈的抑制性。 因此，在拟议的研究中，这种创新的腺病毒表达 方法将用于探测GRK和beta 抑制蛋白参与调节GnRH受体的反应性水平 发信号。该提案的具体目标是：具体目标1： 确定GRK2、GRK3、β抑制蛋白1和2单独和联合应用的作用。 使用腺病毒介导的基因对GnRH刺激的LH分泌的组合 在大鼠垂体细胞中表达。具体目标2：研究消融或 内源性GRK2/3对GnRH受体反应性的中和作用 腺病毒介导的基因转移在垂体中的信号传导 a）肽GRK抑制剂，其竞争性抑制 GRK），和; B）GRK 2/3反义构建体，其抑制GRK的合成， GRKs。具体目标3：确定外部因素（雌二醇，GnRH， 等等）。促性腺激素释放激素受体信号的反应性是通过 调节GRK 2、3和6以及β抑制蛋白的表达水平 1和2.具体目标4：调查GRK的位置和作用方式， 抑制GnRH受体信号传导：a）测量 促性腺激素在致敏和脱敏状态，以确定是否 GRK的作用位点发生在磷脂酶C之前或之后。B）确定 如果感染腺GRK2和β抑制蛋白的促性腺激素细胞的LH分泌 2仍然对NaF处理具有刺激性，NaF是一种直接激活 G蛋白; 3）寻找表位标记的GnRH的磷酸化 受体在表达GRK2/β抑制蛋白2的COS-1细胞中的表达， 标记的血管紧张素II受体作为阳性对照。

The long-term aim of the proposed studies is to investigate regulation of the responsiveness of GnRH receptor signaling. The health importance of these studies is that the mechanism of GnRH's use as a treatment of prostate cancer is based on its propensity to desensitize LH secretion which, in turn, reduced testosterone secretion. Intracellular proteins that have come under scrutiny has potential regulators of the responsiveness of GnRH receptor signaling are members of the GRK family (G protein coupled receptor kinases) and beta arrestins 1 and 2. GRKs 2, 3, and 5 and beta arrestin 1 and 2 are expressed naturally in the rat anterior pituitary gland, and their experimental expression in GnRH receptor expressing COS-1 cells suppresses GnRH-stimulated production of IP3, a member of the second messenger cascade evoke by GnRH. Development of an innovative adenoviral approach for expression of GRKs in rat anterior pituitary cells has shown that they are strongly inhibitory. Therefore, in the proposed studies, this innovative adenoviral expression approach will be used to probe the extent to which the GRKs and beta arrestins participate in setting the responsiveness level of GnRH receptor signaling. The Specific Aims of the proposal are: Specific Aim 1: Determine the effects of GRK2, GRK3, beta arrestin 1 and 2 alone and in combination on GnRH-stimulated LH secretion using adenoviral-mediated gene expression in rat pituitary cells. Specific Aim 2: Investigate ablation or neutralization of endogenous GRK2/3 on responsiveness of GnRH receptor signaling using adenoviral-mediated gene transfer in pituitary gonadotropes of: a) a peptide GRK inhibitor that competitively inhibits GRKs), and; b) a GRK 2/3 antisense construct that suppresses synthesis of GRKs. Specific Aim 3: Determine if the external factors (estradiol, GnRH, etc.) that set responsiveness of GnRH receptor signaling do so by regulating the level of expression of GRK 2, 3, and 6 and beta arrestins 1 and 2. Specific Aim 4: Investigate GRKs' site and mode of action and inhibiting GnRH receptor signaling: a) Measure IP3-responsiveness of gonadotropes during sensitized and desensitized states to determine if GRK's site of action occurs before or after phospholipase C. b) Determine if LH secretion in gonadotropes infected with adeno-GRK2 and beta arrestin 2 remains stimulable to NaF treatment, a reagent that directly activated G proteins; 3) Look for phosphorylation of the epitope-tagged GnRH receptor in GRK2/beta arrestin 2 expressing COS-1 cells using epitope- tagged angiotensin II receptor as a positive control.